Killed or subunit vaccines are often poorly immunogenic, and can result in weak and transient T-cell responses, thus requiring adjuvants to boost the immune response. However, many currently available vaccines include adjuvants that are suboptimal with respect to the quality and magnitude of immune responses they induce. For example, alum, the only approved adjuvant for use in humans in the United States, induces good Th2 type immune responses but is not a potent adjuvant for Th1-type immune responses (HogenEsch et al., Vaccine (2002) 20 Suppl 3:S34-39). Thus, there is a need for additional effective and safer adjuvants.
Two broad categories of adjuvants exist—delivery systems and immunostimulatory adjuvants. Delivery systems include particulate formulations such as liposomes and microparticles. The mechanism of action of these systems are not fully understood but are thought to involve increased uptake by antigen presenting cells (APC) and/or formation of a depot at the site of injection. Immunostimulatory adjuvants stimulate innate immunity resulting in the secretion of cytokines and upregulation of costimulatory molecules. These events are now known to play an instructional role in the development of adaptive immune responses.
The most studied immunostimulatory adjuvants are microbial components, which are potent immune modulating molecules. Bacterial DNA, as well as synthetic CpG oligonucleotides bind to the cellular receptor Toll-Like Receptor 9 (TLR9) and stimulate a cascade of cell signaling events. CpG oligonucleotides are DNA sequences containing an unmethylated CpG dinucleotide, flanked by two 5′ purines and two 3′ pyrimidines. CpG oligonucleotides have been found to stimulate innate immune responses and trigger the production of Th-1 type cytokines, including IFN-γ, IL-6, IL-12, and TNF-α, via interaction of the CpG motif with TLR9 on dendritic cells, macrophages, and B lymphocytes (Klinman et al., Vaccine (1999) 17:19-25; Klinman et al., Proc Natl Acad Sci USA (1996) 93:2879-2883; Krieg, A. M., Nat Rev Drug Discov (2006) 5:471-484; and Krieg et al., Nature (1995) 374:546-549). Co-immunization of protein antigens with synthetic CpG ODNs has been found to increase the production of antigen-specific IgG and direct T-cell responses towards a Th1 phenotype (Ioannou et al., Vaccine (2002) 21:127-137). The adjuvant effects of CpG have been well demonstrated with a variety of viral, bacterial and protozoal antigens in a number of species including mice, cattle, sheep, pigs, and humans (Cooper et al., Aids (2005) 19:1473-1479; Alcon et al., Vaccine (2003) 21:1811-1814; Davis et al., J. Immunol. (1998) 160:870-876; Ioannou et al., J. Virol. (2002) 76:9002-9010; Ioannou et al., Vaccine (2002) 21:127-137).
TLR9 may also bind and be activated by non-CpG DNA (Kindrachuk et al. J. Biol. Chem (2007) 282: 13944-53; Lande et al. Nature (2007) 449:564-9). This activity, coupled with the human cathelicidin LL-37, indicates that the specificity of this receptor may be broadened to microbial or host DNA molecules that are able to localize to the endosome (Lande et al. Nature (2007) 449:564-9).
Viral components such as double stranded (ds) RNA have also been demonstrated to have potent immunostimulatory properties. dsRNA, as well as the synthetic dsRNA analog polyriboinosinic acid-polyribocytidylic acid (poly(I:C)), are recognized by TLR3 resulting in receptor activation (Alexopoulou et al. Nature (2001) 413: 732-8). The expression of TLR3 has been shown to confer responsiveness to purified dsRNA and poly(I:C) in cultured cells. Additionally, TLR3-deficient mice display impaired responses to these ligands (Akira and Takeda Nat Rev Immunol (2004) 4: 499-511). More recently, the host cell component mRNA has been demonstrated to be immunostimulatory due to recognition and activation of TLR3 following release from cells (Kariko et al. J. Biol. Chem. (2004) 26: 12542-12550). TLR3 activation results in the induction of NFkB and IRF3, ultimately leading to the production of antiviral molecules such as type I IFN (Alexopoulou et al., Nature (2001) 413: 732-8). TLR3 activation initiates cascades of phosphorylation and transcriptional activation events that result in the production of numerous inflammatory cytokines that are thought to contribute to innate immunity (Takeda and Akira J. Derm. Sci. (2004) 34:73-82).
Antimicrobial peptides (AMPs), also called “host defense peptides” or “cationic peptides” represent crucial elements of the innate immune system. AMPs can be classified into two broad groups of either cyclic or linear peptides which include a wide variety of molecules such as lysozymes, lactoferrin, secretory leukoprotease inhibitor, defensins and cathelicidins. Typically, AMPs are small molecules which often display a strong cationic charge. AMPs act as effector molecules of innate immunity by killing a broad spectrum of microbes including Gram-positive bacteria, Gram-negative bacteria, fungi, parasites and viruses.
Defensins and cathelicidins are the two major families of mammalian anti-microbial peptides. Defensins display a plethora of immunomodulatory activities, including the ability to stimulate chemotaxis of immature dendritic cells and T-cells, glucocorticoid production, macrophage phagocytosis, mast cell degranulation, complement activation and IL-8 production by epithelial cells (Yang et al., Cell. Mol. Life. Sci. (2001) 58:978-989). Thus, defensins represent an important link between innate and acquired immunity and are potent immune modulators and adjuvants for vaccines. For example, low concentrations of a human α-defensin (10-100 ng, administered with KLH absorbed to alum) lead to strong augmentation of IgG1, IgG2a and IgG2b, indicative of stimulation of both Th1 and Th2 responses (Tani et al., Int. Immunol. (2000) 12:691-700; Lillard et al., Proc. Natl. Acad. Sci. USA (1999) 96:651-656). In contrast, α- and β-defensins, co-delivered intranasally, have been reported to stimulate primarily a Th-2 response (IgG1 and IgG2b, but not IgG2a or IgM) to ovalbumin (Brogden et al., Int. J. Antimicrob. Agents (2003) 22:465-478). Intradermal immunization of mice with a fusion construct encoding the HIV glycoprotein 120 and β-defensin 2 resulted in strong humoral and cell-mediated mucosal immune responses against HIV and antitumor immune responses were greatly enhanced by the presence of defensins.
Likewise, cathelicidins, another class of endogenous mammalian host defense peptides, have been found to exert a number of immune-modulating functions. Besides their well-documented antimicrobial activity, cathelicidins act as chemotactic factors, induce cytokine and chemokine expression, alter gene expression in host cells, and modulate dendritic cell function (Bowdish et al., Antimicrob. Agents Chemother. (2005) 49:1727-1732; Brown et al., Curr. Opin. Immunol. (2006) 18:24-30; Hancock, R. E., Lancet Infect. Dis. (2001) 1:156-164). Recent evidence has also shown that the human cathelicidin LL-37 (An et al., Leuk. Res. (2005) 29:535-543) and mouse cathelin-related antimicrobial peptide (CRAMP) (Kurosaka et al., J. Immunol. (2005) 174:6257-6265) were able to enhance adaptive immune responses.
Indolicidin, one of the smallest known host defense peptides, is a linear 13-amino acid peptide found in the cytoplasmic granules of bovine neutrophils (Selsted et al., J. Biol. Chem. (1992) 267:4292-4295). In vitro it was found to inhibit LPS-induced TNF-α secretion by human macrophage-like cells, and induce production of the chemokine IL-8 in human bronchial epithelial cells (Bowdish et al., Antimicrob. Agents Chemother. (2005) 49:1727-1732), however its activity as an adjuvant in vivo has yet to be established.
Polyphosphazenes are high-molecular weight, water-soluble polymers, containing a backbone of alternating phosphorous and nitrogen atoms (Payne et al., Vaccine (1998) 16:92-98). One of the most investigated polyphosphazene polyelectrolytes, poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) has been found to exert adjuvant activity when incorporated into a number of vaccine formulations, including influenza (Payne et al., Vaccine (1998) 16:92-98), human rotavirus (McNeal et al., Vaccine (1999) 17:1573-1580), and cholera vaccines (Wu et al., Infect. Immun. (2001) 69:7695-7702). Similarly, poly(di-4-oxyphenylproprionate)phosphazene (PCEP) has been shown to enhance antigen-specific immune responses to influenza antigens (Mutwiri et al., Vaccine (2007) 25:1204-1213). Polyphosphazene adjuvant activity does not appear to be due to the formation of an injection-site depot, but rather may be linked to the ability of the polymer to form water-soluble, non-covalent complexes with antigens, stabilizing them and allowing efficient presentation to immune cells (Andrianov et al., Biomacromolecules (2005) 5:1999-2006; Payne et al., Adv. Drug Deliv. Rev. (1998) 31:185-196).
Co-administration of PCPP with a CpG oligonucleotide has been shown to enhance immune responses in mice immunized with hepatitis B surface antigen (Mutwiri et al., Vaccine (2008) 26:2680-2688). Additionally, intranasal immunization using a formalin-inactivated bovine respiratory syncytial virus (BRSV) vaccine co-formulated with a CpG oligonucleotide and PCPP resulted in enhanced protection against BRSV challenge (Mapletoft et al., J. Gen. Virol. (2008) 89:250-260).
Despite the various advances in adjuvant technology, there remains a need for safe and effective methods to prevent infectious diseases. Thus, the wide-spread availability of new adjuvants would be highly desirable and could save a considerable number of lives.